In freshly isolated rabbit SANC, the PP1/2A inhibitor calyculin A (CyA), 100-500 nM, increased the spontaneous SANC beating rate, recorded using perforated patch, by 24-30%, from 179.413.1 to 230.67.45 beat/min (n=21). Confocal Ca2+ imaging of isolated SANC demonstrated that PP inhibition significantly increased LCR size from 5.50.5 to 8.11.3 um (n=12), and decreased the LCR period from 304.815.9 to 242.313.2 ms (n=12). The decrease in the LCR period predicted the reduction in the spontaneous SANC cycle length (CL) from 390.317.1 to 314.414.5 ms, R squared=84 (n=12). Assessment of PLB phosphorylation at PKA-dependent and CaMKII-dependent sites using phosphorylation site-specific polyclonal antibodies indicated that basal phosphorylation at Ser16 and Thr17 was increased by 1.65- and 3.34-fold after incubation with 100 nM CyA for 10 min (n=5, 4). Okadaic acid, at 1-100 nM, a concentration specific for the inhibition of PP2A, did not have a significant effect on the spontaneous AP, CL, LCRs parameters and PLB phosphorylation indicating that PP1 rather than PP2A is involved in regulation of spontaneous rhythmic beating in SANC. Biochemical measurement showed a high level of PP activity which was inhibited by CyA. Thus basal PLB phosphorylation in SANC is determined by the balanced activity of phosphatases and kinases. We show, for the first time, that basal PP1 activity regulates spontaneous SANC AP firing rate and modulates SR generated LCRs via its effect on PLB phosphorylation, while PP2A is not involved in this regulation.